The ends of linear chromosomes are unique structures that require special handling by the cell. If left unattended, these ends are inappropriately processed impacting both genomic stability and cellular proliferation. Telomeres, the specialized nucleoprotein structures that cap and protect the ends of chromosomes, restore chromosomal stability and allow continued proliferation. These functions are mediated by the action of the Pot1 (Protection of Telomeres 1) family of proteins, which targets the 3' single-stranded overhang region of the telomere via a specialized single-stranded DNA binding domain (DBD). Pot1 is required for normal cellular proliferation in the model organisms S. pombe and the mouse, playing an integral role in telomere maintenance by providing a capping function as well as regulating the action of telomerase at the telomere. However, our understanding of human Pot1 function in vivo is less clear, primarily due to the caveats associated with RNAi/shRNA and transient transfection approaches. Therefore, a novel method for targeting Pot1 would greatly facilitate mechanistic studies of Pot1 function at the telomere. Association of Pot1 with telomeric ssDNA is required for its function. One unexplored method for studying Pot1 function is a "chemical genetics" approach that uses small molecule inhibitors that target its ssDNA-binding activity. Examination of high-resolution structures bound to ssDNA reveal a protein/DNA interface that is chemically rich and forms a detailed and unique surface, suggesting that a small molecule could specifically disrupt this interaction. We propose to develop and implement a high-throughput screening (HTS) assay to identify small molecules that target the ssDNA-binding activity of Pot1 proteins from S. pombe and humans. In our first specific aim, we will devise and validate a time-resolved FRET-based assay targeting Pot1/DNA binding. This "mix and measure" type assay is highly sensitive, robust, and reproducible, making it ideal for a successful HTS study. In the second specific aim, we will configure the assay for entry into the HTS format by testing two chemically distinct pilot libraries. A strategy for validation and prioritization of hit compounds is presented. Finally, we propose a series of follow up studies with "hit" compounds from the pilot screening to (a) characterize the mode and mechanism of ssDNA-binding inhibition using biochemical and structural tools and (b) study their impact on proper telomere function in vivo. Our proposed research program will be greatly aided through a collaboration with Prof. Charles McHenry, a leader in the field who uses HTS to identify inhibitors of bacterial replication and has the tools and expertise in place to assist with the design and implementation of the screen. Telomeres play key roles in cancer and aging due to their roles in discriminating natural DNA ends from damaged DNA and compensating for the inability of the standard replication machinery to fully copy the chromosomal terminus. As a result of these activities, proper telomere function is integral to human health. Small molecules that specifically target telomeres will provide valuable tools for dissecting the mechanism of telomere function and lay the groundwork for the discovery of cancer therapeutics. [unreadable]